Ubiquitin, a small and compact α+β protein without any disulfide
linkages, mediates intracellular protein degradation and many important
biological functions.The formation of a β turn in the N-terminal β-hairpin has been
implicated in the transition state of ubiquitin folding pathway.From earlier studies, we have shown
that the Gly-10 residue is important in the stability of the hairpin.Deleting Gly-10 destabilizes the β-hairpin
structure.On the contrary,
mutating Thr-9 to Asp-9 has the opposite effect.Phe-4, a residue on the strand of the hairpin that
participates as part of a small exterior hydrophobic cluster, also appears to
contribute the stability of the hairpin.Here, we compare the effects of these residues in the folding of
ubiquitin by examining the refolding/unfolding kinetics of the mutated
proteins: desG10/F45W, T9D/F45W, and F4A/F45W.F45W mutation was introduced in order to provide a good
fluorescence probe.Kinetic
refolding and unfolding studies of these mutants were monitored by stopped-flow
circular dichroism and stopped-flow fluorescence spectroscopy. From the results obtained, we propose
that the turn residues are more important than the strand residue in the
formation of the transition state.